Adorbent for eliminating hepatitis C virus, adsorber, and adsorption method

ABSTRACT

An adsorbent for removing hepatitis C virus which has the ability to adsorb HCV particles, particularly immune-complex HCV particles, from a patient&#39;s body blood safely and with high efficiency and high selectivity for enhancing the efficacy of interferon therapy, an HCV adsorption apparatus including said adsorbent, and a adsorbing method for removing HCV are provided.  
     An adsorbent for removing hepatitis C virus which comprises a compound capable of adsorbing hepatitis C virus as immobilized on a water-insoluble carrier, an adsorption apparatus including said adsorbent, and an adsorbing method for removing HCV.

TECHNICAL FIELD

[0001] The present invention relates to an adsorbent for removinghepatitis C virus which is capable of selectively adsorbing hepatitis Cvirus from body fluids such as blood, plasma, etc. to thereby expeditethe cure for hepatitis C, an adsorption apparatus including saidadsorbent, and an adsorbing method for removing hepatitis C virus.

PRIOR ART

[0002] With the successful cloning of the RNA virus genome of hepatitisC virus in 1989 (Q. L. Choo et al.: Science, 244, 359, 1989), it becamepossible to assay anti-hepatitis C virus antibody using a recombinantprotein. As a result, most of the hepatitis termed non-A, non-Bhepatitis in the past were found to be hepatitis C. Thus, it isestimated that in Japan today there are about 2,000,000 HCV carriersand, of them, 1,400,000 have chronic hepatitis and 300,000 havecirrhosis (Shiro Iino: Medical Practice in Gastroenterology-2, HepatitisC, 11-17, 1993).

[0003] According to the Ministry of Health and welfare demographicstatistics, the number of deaths due to primary liver cancer in 1992 was27 thousand (1992 Demographic Statistics, Minister of Health and WelfareStatistical Information Bureau, Vol. 1, 1993) and approximately 70% ofthe casualties were due to hepatocellular carcinoma associated withhepatitis C virus infection and it is by now considered that this cancerensues following the progression of chronic hepatitis to cirrhosis (S.Kaneko et al.: Intervirology, 37, 108, 1994; Eiki Matsushita et al.:Japanese Journal of Clinics, 53, 727, 1995 Special Issue). Therefore,hepatitis C can be said to be a refractory disease which progresses tocirrhosis to hepatocellular carcinoma.

[0004] The conventional therapy of hepatitis C is mostly built aroundrest cure, dietary thereby, and pharmacotherapy using hepatoprotectantsand/or Chinese medicines. However, because the hepatitis virus cannot beremoved by such therapeutics, the cure rate is miserably low. This iswhy, in clinical practice, emphasis has been placed on the arrest ofprogression of chronic liver disease through palliation of local tissuenecrosis. Therefore, as the disease period is prolonged, many patientssuccumb to hepatocellular carcinoma, the serious outcome, throughcirrhosis as mentioned above.

[0005] Meanwhile, the mass production of interferons became feasiblerecently and those proteins were found to show not only antiviralactivity against hepatitis C virus and its cognate RNA viruses in vitro(Yasuyuki Ninomiya et al.: The Clinical Report, 19, 23i, 1985) but alsoprotective activity in mice infected with RNA viruses (M. Kramer et al.:J. Interferon Res., 3, 425, 1983). Accordingly, the utility ofinterferons in clinical cases of hepatitis C has come into the focus ofattention.

[0006] Actually, serum transaminase was normalized in some of the non-A,non-B hepatitis cases which were treated with a recombinantinterferon-alpha (J. H. Hoofnagel et al.: N. Eng. J. Med., 315, 1575,1986) and in the administration of an interferon to patients withhepatitis C, some cases became consistently negative to hepatitis Cvirus RNA in blood (K. Chayama et al.: Hepatology, 13, 1040, 1991;Hideki Ogiwara et al.: Japanese Journal of Gastroenterology, 88, 1420,1991). In view of those results, interferons have come to be broadlyused in clinical practice. Thus, the therapy of hepatitis C has made adecisive step forward from symptomatic therapy to etiotropic therapy.

[0007] However, in the interferon therapies performed in about 200,000cases of type C chronic active hepatitis during the past several yearsin Japan, it was only in about 30% of cases that the virus could beeliminated and the disease cured and in the remaining about 70% of casesthe virus survived and the therapy either proved ineffective orrecurrences were encountered (Migito Yano: Japanese Journal of Clinics,53, 986, 1995 Special Issue).

[0008] In the success or failure of a therapy, the hepatitis C virusgene type, the viral population in blood, and the stage of liver diseaseare important factors but, of all the factors involved, the viralpopulation in blood is the most important factor. For example, when theamount of the virus in 1 ml of the patient's blood was less than1,000,000 copies, the virus could be eliminated from the body and thedisease cured by administration of an interferon in about 80% of casesbut when the amount of the virus was over 1,000,000 copies, the curerate was as low as about 9% (Fumio Imazeki et al.: Japanese Journal ofClinics, 53, 1017, 1995).

[0009] In addition to the above-mentioned amount of the virus, theinventors of the present invention found that the mode of existence ofviral particles in blood is also an important factor modifying theeffect of an interferon therapy. Thus, it has been reported thathepatitis C virus particles in blood can be classified according totheir suspension density in blood into low-density particles with highinfectivity and high-density particles with low infectivity. Therefore,the inventors studied the relationship of those viral particles varyingin density to the severity of illness and the interferon therapy andfound that whereas the interferon therapy resulted in cure in 75% ofpatients with the ratio of low-density viral particles to high-densityviral particles is 10:1, the cure rate in patients with the ratio of1:10 was as low as 13%.

[0010] It was also found that in blood, the low-density virus particlesis bound to lipoprotein and the high-density virus particles toimmunoglobulin, thus existing as immune complexes (Akihito Sakai et al.:Japanese Journal of Gastroenterology, 92 (Special Issue), 1488, 1995).

[0011] It is, therefore, clear that the contemporary interferon therapyhas the drawback that the lower the blood viral population is or thelower the immune-complex virus population is, the higher is thetherapeutic response and conversely the higher the viral population isor the hither the immune-complex virus population is, the much lower isthe therapeutic response.

SUMMARY OF THE INVENTION

[0012] The present invention has for its object to provide an adsorbentfor removing hepatitis C virus which has the ability to adsorb hepatitisC virus particles, particularly immune-complex hepatitis C virusparticles, from a patient's body blood safely and with high efficiencyand high selectivity for enhancing the efficacy of interferon therapy,an hepatitis C virus adsorption apparatus including said adsorbent, andan adsorbing method for removing hepatitis C virus.

[0013] For accomplishing the above object, the inventors of the presentinvention made an intensive exploration for a compound which, whenimmobilized on a water-insoluble carrier and brought into contact with apatient's blood, should exhibit a high adsorbing affinity for hepatitisC virus but not for such proteins as albumin. As a result, the inventorsfound that an adsorbent fabricated by immobilizing a compound capable ofadsorbing hepatitis C virus, particularly a compound having a bindingaffinity for immunoglobulin and/or immune complex, on a water-insolublecarrier displays are markably high hepatitis C virus-adsorbingperformance. The present invention has been developed on the basis ofthe above finding.

[0014] The present invention, therefore, is directed to an adsorbent forremoving hepatitis C virus which comprises a compound capable ofadsorbing hepatitis C virus as immobilized on a water-insoluble carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a diagrammatic representation of the relation betweenflow rate and pressure loss for glass columns packed with variouswater-insoluble carriers. The ordinate represents flow rate (cm/min.)and the abscissa represents pressure loss (kg/cm²);

[0016]FIG. 2 is a schematic cross-section view of the hepatitis C virusadsorption apparatus according to the invention; and

[0017]FIG. 3 is a diagram showing the pUCNTMK3P47 vector.

[0018] Each numeric symbol represents in the following.

[0019]1. outlet

[0020]2. inlet

[0021]3. absorbent for removing

[0022]4,5. means for preventing leakage of the absorbent

[0023]6. column

[0024]7. apparatus

DETAILED DESCRIPTION OF THE INVENTION

[0025] Hereinafter, the present invention is described in detail.

[0026] The adsorbent for removing hepatitis C virus according to thepresent invention comprises a water-insoluble carrier and, asimmobilized thereon, a compound capable of adsorbing hepatitis C virus.

[0027] The compound capable of adsorbing HCV is not particularlyrestricted only if it adsorbs hepatitis C virus but the preferredcompound has a binding affinity for immunoglobulin and/or immunecomplex.

[0028] The preferred compound, among such compounds capable of adsorbinghepatitis C virus coupled to immunoglobulin and/or immune complex, is acompound which may preferentially and efficiently adsorb hepatitis Cvirus coupled to immunoglobulin and/or immune complex in comparison withhepatitis C virus particles as such.

[0029] More preferably, the above-mentioned compound having a bindingaffinity for immunoglobulin and/or immune complex is animmunoglobulin-binding protein.

[0030] The immunoglobulin-binding protein includes but is not limited toprotein A, protein G, protein H, protein L, protein M, rheumatoidfactor, and complement.

[0031] Still more preferably, the compound having a binding affinity forimmunoglobulin and/or immunoglobulin complex is an anti-immunoglobulinantibody.

[0032] Even more preferably, the above-mentioned compound capable ofadsorbing hepatitis C virus is a component of saidimmunoglobulin-binding protein and/or anti-immunoglobulin antibody,which component is a fragment protein or a peptide containing a bindingsite for immunoglobulin and/or immune complex or a derivative of saidprotein fragment or peptide.

[0033] Referring to the water-insoluble carrier, a porous carrier is apreferred example.

[0034] Preferably, the porous water-insoluble carrier has a mean porediameter of from 10 to 1500 nm.

[0035] Another preferred form of the water-insoluble carrier is asubstantially nonporous carrier. Moreover, the water-insoluble carrieris preferably hydrophilic.

[0036] The adsorbent for removing hepatitis C virus according to thepresent invention can be used for the purpose of removing hepatitis Cvirus from body fluids inclusive of blood and plasma.

[0037] The adsorbent for removing hepatitis C virus can also be used forthe purpose of removing immune complex forms of hepatitis C virus frombody fluids inclusive of blood and plasma.

[0038] The hepatitis C virus adsorption apparatus according to thepresent invention comprises a casing having an inlet and an outlet foradmission and discharge of a fluid and housing any of theabove-mentioned adsorbents and a means for preventing leakage of saidhepatitis C virus adsorbent from the casing.

[0039] The adsorbing method for removing hepatitis C virus comprises astep of contacting any of said adsorbents with a body fluid containinghepatitis C virus.

[0040] The HCV-containing body fluid for use in the method for removingHCV according to the present invention includes blood and plasma, amongother body fluids.

[0041] The preferred embodiments of the invention are now described,although the invention is not limited to those specific embodiments.

[0042] The compound capable of adsorbing hepatitis C virus used in thepresent invention is a compound which is capable of adsorbing hepatitisC virus in a substantial measure and, as such, is not limited in kind.Preferably, however, it is a substance which may specifically bind theheavy chain or light chain of immunoglobulin and/or immunoglobulincomplex.

[0043] The above-mentioned substance capable of binding the heavy chainor light chain of immunoglobulin and/or immune complex specificallyincludes the compounds which are generally called immunoglobulin-bindingproteins such as protein A, protein G, protein H, protein M, rheumatoidfactor, and complement, which can bind the Fc domain in the heavy chainof immunoglobulin G, and protein L which has a binding affinity for thelight chain (L. Bjorck: J. Immunol., 140, 1194, 1988; H. Gomi et al.: J.Immunol., 144, 4046, 1990; Hisayuki Doi: Meneki Rinsho (ClinicalImmunology), 23, 896, 1991) and anti-immunoglobulin antibodies.

[0044] There can also be mentioned those fragments of theabove-mentioned substances which have a substantial binding affinity forimmunoglobulin and/or immune complex, for example the peptidescorresponding to the 58-residue A through E domains, which areimmunoglobulin-binding sites, of proteinA (M. Uhlen et al.: J. Biol,Chem. 259, 1965, 1984), the FB29 peptide which is a further abridgementof the B domain peptide of protein A (J. S. Huston et al.: BiophysicalJ., 62, 87, 1992), the peptides corresponding to the 55-residue C1-C3domains of protein G (B. Guss et al.: EMBO J., 5, 1567, 1986), the Adomain peptide of protein H (H. Gomi et al.: ibid), the B1-B5 domainpeptides of protein L (Bjorck, Laruth, et al., Japanese KohyoPublication Hei-7-506573), and the CBP2 peptide of complement Clq (M. A.Baumann et al.: J. Biol. Chem., 265, 18414 (1990), etc., and otherimmunoglobulin-binding domain peptides of so-calledimmunoglobulin-binding proteins, and their derivatives.

[0045] Furthermore, the Fab and F(ab)₂ fragments of rheumatoid factor oranti-immunoglobulin antibody, single-strand Fv polypeptide, etc. canalso be mentioned as representative examples.

[0046] The water-insoluble carrier which can be used in the presentinvention is not particularly restricted but includes inorganic carrierssuch as glass beads, silica gel, etc., organic carriers such assynthetic polymers, e.g. crosslinked polyvinyl alcohol, crosslinkedpolyacrylate, crosslinked polyacrylamide, crosslinked polystyrene, etc.and polysaccharides such as crystalline cellulose, crosslinkedcellulose, crosslinked agarose, crosslinked dextran, etc., andorganic-organic or organic-inorganic composite carriers consisting ofsuch materials.

[0047] Particularly preferred are hydrophilic carriers, for suchcarriers are characterized in that the amount of non-specific adsorptionis relatively small and the adsorption selectivity to hepatitis C virusis high. The term “hydrophilic carrier” is used herein to mean a carrierthe constituent compound of which has a water-contact angle of notgreater than 60 degrees when it is molded in a flat sheet form.

[0048] The carrier of this kind is not particularly restricted butincludes carriers made of polysaccharides such as cellulose, chitosan,Sepharose, dextran, etc., polyvinyl alcohol, saponified ethylene-vinylacetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylicacid, poly(methyl methacrylate), polyacrylic acid-polyethylene alloy,polyacrylamide-polyethylene alloy, glass, and so forth.

[0049] Particularly, carriers containing OH groups are superior inadsorptive capacity and selectivity. Moreover, porous cellulose gel hasthe following advantageous features (1)-(4) and, as such, is one of themost preferred carriers for use as the water-insoluble carrier in thepractice of the invention.

[0050] (1) Because of its comparatively high mechanical strength andtoughness, this gel is not easily disintegrated into dust-like fineparticles by stirring, etc. and, when packed into a column, it is notappreciably compacted or plugged even when a body fluid is passedthrough the bed at a high flow rate. Moreover, because of the porousstructure, it is not liable to undergo dimensional change even whensterilized by autoclaving.

[0051] (2) Because it is made of cellulose, the gel is hydrophilic witha large number of hydroxyl groups available for binding to the ligandand is low in nonspecific adsorption.

[0052] (3) Since a comparatively high strength can be retained even ifthe pore volume is increased, it may have an adsorptive capacity aslarge as a soft gel.

[0053] (4) Compared with synthetic polymer gel or other gels, this gelis higher in safety.

[0054] The water-insoluble carriers mentioned above can be used eachalone or as a suitable mixture of two or more species.

[0055] In consideration of its application as an adsorbent for removinghepatitis C virus and the mode of use, the water-insoluble carrier foruse in the present invention preferably has a large surface area and, inthis sense, is preferably a carrier having a large number of pores,namely porous.

[0056] The preferred mean pore diameter of said porous water-insolublecarrier is between 10 and 1500 nm. Hepatitis C virus particles are 50-55nm in diameter and in order that such virus particles may be efficientlyadsorbed with a porous carrier, the pore size distribution profile ofthe carrier is preferably biased far toward the range larger than thediameter of virus particles. If the pore diameter is too large, thestrength of the carrier will be sacrificed and the surface areadecreased. The still more preferred mean particle diameter is between 50and 1250 nm.

[0057] On the other hand, the virus can be adsorbed even with a carrierwhich is substantially nonporous. This kind of carrier can also beutilized for exploiting the advantage that the proteins and othercomponents useful for the body in the body fluid (blood, plasma, serum,etc.) are little adsorbed in the substantial absence of pores.

[0058] The term “substantially nonporous” is used in this specificationto include porous carriers having very small pores (e.g. less than 10nm).

[0059] Referring, further, to the porous structure of saidwater-insoluble carrier, it is preferable, in view of the adsorptivecapacity per unit volume of the adsorbent, that the pores should not beconfined to the surface but be distributed throughout the carrier andthat the carrier has a fractional pore volume of not less than 20% and aspecific surface area of not less than 3 m²/g.

[0060] The form of said water-insoluble carrier is not particularlyrestricted but includes bead-form, fibrous form, and film form(inclusive of hollow fiber), among others. From the standpoint ofhydrodynamics of the body fluid in extracorporeal circulation, abead-form carrier is preferably used. As to the mean particle diameterof said bead-form carrier, beads within the range of 10 and 2500 μm areeasy to use. However, it is preferable to use beads in the range of 25and 800 μm.

[0061] The existence of functional groups available for immobilizationof the ligand on the surface of the water-insoluble carrier isbeneficial to immobilization.

[0062] The representative examples of such functional groups includehydroxyl, amino, aldehyde, carboxyl, thiol, silanol, amido, epoxy,succinylimido, acid anhydride, and other functional groups.

[0063] The above-mentioned water-insoluble carrier may be either a rigidgel and a soft gel. For the application as an adsorbent forextracorporeal circulation therapies, it is important that, when packedin a column, there should not occur plugging, i.e. clogging of pores, onpassage of a body fluid. Therefore, to insure a sufficient mechanicalstrength, the water-insoluble carrier is preferably a rigid carrier.

[0064] In the context of the present invention, the rigid carrier is acarrier such that when the gel, in the form of e.g. beads, is uniformlypacked into a glass cylindrical column (9 mm in. dia., 150 mm long)under the following conditions and an aqueous fluid is passed throughthe packed column, a linear relationship is obtained between pressureloss (AP) and flow rate up to 0.3 kg/cm².

[0065] For example, a glass cylindrical column (9 mm in. dia., 150 mmlong) fitted with a 15 μm (pore diameter) filter at either end wasuniformly packed with an agarose gel (Bio-Rad, Biogel-ASm, particlediameter 50-100 mesh), a vinyl polymer gel (Toyo Soda, Toyopearl HW-65,particle diameter 50-1000 μm), or a cellulose gel (Chisso Corporation,Cellulofine GC-700m, particle diameter 45-105 μm), water was introducedinto the column using a peristatic pump, and the relationship betweenflow rate and pressure loss (A P) was plotted (FIG. 1).

[0066] The flow rate (cm/min) was plotted on the ordinate and thepressure loss (kg/cm²) was plotted on the abscissa. In FIG. 1, ◯represents Toyopearl HW-65, Δ represents Cellulofine GC-700m, andrepresents Biogel-A5m.

[0067] As a result, whereas the flow rate increased in approximateproportion with an increase in pressure in the case of Toyopearl HW-65and Cellulofine GC-700m, Biogel-A5m underwent compaction so that theflow rate could not be increased by raising the pressure.

[0068] In the immobilization of an immunoglobulin-binding protein orpeptide on the water-insoluble carrier in accordance with the presentinvention, the immobilization is preferably effected through ahydrophilic spacer in order to reduce the steric hindrance of theprotein or peptide for improving the adsorption efficiency and suppressnon-specific adsorption.

[0069] The preferred hydrophilic spacer may for example be apolyalkylene oxide derivative available upon modification of thepolyalkylene chain with a substituent group such as carboxyl, amino,aldehyde, or epoxy at either end.

[0070] The compound having a binding affinity for immunoglobulin and/orimmune complex which is to be immobilized on said water-insolublecarrier and the organic compound as a spacer can be immobilized by anysuitable technique. Among such techniques are those which areconventionally used in the immobilization of proteins and peptides oncarriers, such as the methods utilizing the epoxy reaction, Nic basereaction, condensation reaction using a carbodiimide reagent, activeester reaction, and carrier crosslinking reaction using glutaraldehydereagent.

[0071] In consideration of the fact that the adsorbent for removinghepatitis C virus according to the invention is an adsorbent chieflyused in extracorporeal circulation therapy and hemocatharsis, it ispreferable to use an immobilization method which insures that in thesterilization of the adsorbent and during such therapy, the proteinsetc. will not readily be released out from the water-insoluble carrier.For example, the following methods can be mentioned.

[0072] (1) The method which comprises reacting the carboxyl group of thecarrier with N-hydroxysuccinimide to substitute a succinimidoxycarbonylgroup and causing it to react with the amino group of the protein orpeptide (active ester method).

[0073] (2) The method which comprises subjecting the amino or carboxylgroup of the carrier to condensation reaction with the carboxyl or aminogroup of the protein or peptide in the presence of a condensing agentsuch as dicyclohexylcarbodiimide (condensation method).

[0074] (3) The method in which the protein or peptide is crosslinkedusing a compound having two or more functional groups, such asglutaraldehyde (carrier crosslinking method).

[0075] For suppressing the release and elution of the protein from thecarrier, immobilization is preferably effected by covalent bonding.

[0076] The adsorbing method for removing hepatitis C virus from a bodyfluid by contacting a carrier carrying a compound capable of adsorbinghepatitis C virus with a body fluid such as blood, plasma or serum canbe practiced in a variety of manners. Specifically, for example, thefollowing methods can be mentioned.

[0077] (1) The method which comprises withdrawing a body fluid from thepatient's body, pooling it in a bag or the like, mixing the adsorbentwith the body fluid there to remove hepatitis C virus, filtering off theadsorbent and returning the substantially hepatitis C virus—free bodyfluid to the body.

[0078] (2) The method which comprises providing a casing having an inletand an outlet and fitted with a filter permeable to the body fluid butnot to the adsorbent across said outlet, packing the casing with theadsorbent, and passing the body fluid through the packed adsorbent.

[0079] Although both methods can be selectively employed, the secondmethod (2) is expedient and simple procedure-wise. Moreover, when saidcasing or column is built into a tubing system for extracorporealcirculation, hepatitis C virus can be efficiently and directlyeliminated from the patient's body fluid. The adsorbent for removinghepatitis C virus according to the invention is suited for the lattermethod.

[0080] The hepatitis C virus adsorption apparatus including theadsorbent for removing hepatitis C virus according to the invention isnow described with reference to the accompanying schematic drawing.

[0081] Referring to FIG. 2, the apparatus 7 has a liquid inlet or outlet1, a liquid outlet or inlet 2, the hepatitis C virus adsorbent of theinvention 3, means 4 and 5 for preventing leakage of the adsorbent,through which the body fluid and its components may pass freely but theadsorbent cannot pass, and a column 6.

[0082] The geometry and material of the apparatus are not particularlyrestricted. However, it is preferable to use a cylindrical apparatushaving a capacity of about 20-400 mL and a diameter of about 2-10 cm.

BEST MODE FOR CARRYING OUT THE INVENTION

[0083] The following examples illustrate the present invention infurther detail without delimiting its metes and bounds. In thisspecification, various amino acid residues are indicated by thefollowing abbreviations.

[0084] Ala: L-alanine residue, Asp: L-aspartic acid residue, Asn:L-asparagine residue, Cys: L-cysteine residue, Gln: L-glutamine residue,Glu: L-glutamic acid residue, Gly: L-glycine residue, Ile: L-isoleucineresidue, Leu: L-leucine residue, Lys: L-lysine residue, Phe:L-phenylalanine residue, Thr: L-threonine residue, Trp: L-tryptophanresidue, Tyr: L-tyrosine residue, Val: L-valine residue.

[0085] In this specification, the amino acid sequence of a peptide isdescribed in the conventional manner, assuming that its amino terminal(hereinafter referred to as N-terminus) is situated at the left end andits carboxyl terminal (referred to as C-terminus) at the right end.

EXAMPLE 1

[0086] Immobilization of an IgG-Binding Protein (Protein A) on a PorousCarrier (GCL 2000m)

[0087] Expoxy Activation of Cellulose Gel

[0088] The cellulosic porous rigid gel GCL-2000m (Chisso Corporation,globular protein cutoff molecular weight 3×10⁶), 90 mL, was made up withwater to 180 mL, then 60 mL of 2 M sodium hydroxide was added, and thegel temperature was adjusted to 40° C. To this gel was added 21 mL ofepichlorohydrin, and the epoxidation reaction was carried out at 40° C.for 1 hour. After completion of the reaction, the gel was thoroughlyrinsed with water to provide an epoxy-activated cellulose gel.

[0089] Immobilization of Protein A

[0090] In 0.5 mL of 0.05 M borate buffer (pH 10.0) was dissolved 4 mg ofprotein A (Sigma), and 0.01 N NaOH/water was added so as to bring the pHto 10 and make a total volume of 1.0 mL (protein A solution). Thisprotein solution (total amount) was added to 1 mL of the aboveepoxy-activated cellulose gel and the mixture was shaken at 37° C. for16 hours and washed with a sufficient amount of PBS (10 mM phosphatebuffer supplemented with 150 mM sodium chloride) to provide GCL2000m-Protein A.

[0091] Quantitation of the Immobilized Protein

[0092] The protein A concentration was measured in the reaction mixtureby HPLC before and after the immobilization reaction and the reactionrate was calculated to find the amount of immobilization. It was foundthat 2.1 mg of protein A was immobilized per mL of Protein A-GCL2000m.

EXAMPLE 2

[0093] Immobilization of an IgG-Binding Protein (Protein G) on a PorousCarrier (GCL2000m)

[0094] Using protein G (Pharmacia LKB) in lieu of protein A, theprocedure of Example 1 was otherwise repeated to provideGCL2000m-Protein G (3.2 mg/mL).

EXAMPLE 3

[0095] Immobilization of the IaG-Binding Domain of Protein G on a PorousCarrier (Sepharose 6B)

[0096] Synthesis of a Peptide

[0097] A peptide having the amino acid sequence of 57 residues in the C3domain of protein G with cysteine added to the N-terminus wassynthesized by the solid-phase method using Peptide Synthesizer Model4170 (Pharmacia LKB).

[0098] Using 0.1 mmol of Fmoc-glutamine NovaSyn KA, a resin carrying theC-terminal glutamine, the deprotection reaction and condensationreaction were repeated in the direction toward the N-terminus forpeptide chain extension in accordance with the input program of theabove peptide synthesizer.

[0099] Thus, the cycle of removal of the α-amino-protecting group, i.e.9-fluorenylmethyloxycarbonyl (Fmoc), from the amino acid withpiperidine, washing with dimethylformamide (DMF), the condensationreaction using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate and diisopropylethylamine, and subsequent washing withDMF was repeated.

[0100] The amino acids were used in the forms of Fmoc-L-Ala,Fmoc-L-Asn(Trt), Fmoc-L-Asp(OtBu), Fmoc-L-Cys(Trt), Fmoc-L-Gln(Trt),Fmoc-L-Glu(OtBu), Fmoc-L-Gly, Fmoc-L-Ile, Fmoc-L-Leu, Fmoc-L-Lys(Boc),Fmoc-L-Phe, Fmoc-L-Thr(tBu), Fmoc-L-Trp, Fmoc-L-Tyr (tBu), andFmoc-L-Val and each in an amount of about 5 molar equivalents (0.5 mmol)based on the substrate in the vial. Here, Trt, OtBu, Boc, and tBurepresent trityl, tert-butyl ester, tert-butyloxycarbonyl, andtert-butyl, respectively. Deprotection and cleavage of the peptide chain

[0101] After completion of the reaction series for all the amino acids,the carrier was washed successively with tert-amyl alcohol, acetic acid,and diethyl ether on a 3G-3 pore glass filter and, then, dried in vacuoto provide a dry carrier. To 1 g of the obtained carrier in the vial, 20mL of trifluoroacetic acid (TFA), 260 μL of 1,2-ethanedithiol, and 780μL of anisole were added and the mixture was stirred at room temperaturefor 1.5 hours.

[0102] Then, this mixture was filtered through a 3G-3 pore glass filterto remove the carrier and the filtrate was concentrated under reducedpressure at a temperature of 35° C. To the residue was added anhydrousdiethyl ether cooled ahead of time until a precipitate ceased to appearunder stirring, followed by centrifugation, and the crude peptide pelletwas recovered. This crude peptide was rinsed with several portions ofanhydrous diethyl ether and dried in vacuo to provide the objectivecrude peptide.

[0103] Purification of the Peptide

[0104] The above crude peptide was dissolved in 0.1% TFA and filteredthrough a 0.2 μm membrane filter and the filtrate was subjected to highperformance liquid chromatography. For this HPLC, Model LC-10A System(Shimadzu) and, as the column, μ Bondasphere C18 (NipponMillipore-Waters) were used in a reversed phase. As the mobile phase,0.1% TFA/H₂O was used as solvent A and 0.1% TFA-80% (v/v)acetonitrile/H₂O for solvent B and elution was carried out on a lineargradient from Solvent A to solvent B.

[0105] The fraction corresponding to a chromatographic peak wascollected. Fractional elution was repeated several times and the pooledfraction was lyophilized to provide a purified peptide. This peptide wassubjected to amino acid analysis using Gas-phase Protein Sequencer 477(Applied Biosystems) and Hitachi Custom Ion Exchange Resin to confirmthat the peptide obtained had the amino acid sequence shown in SEQ IDNO:1.

[0106] Immobilization of the Peptide

[0107] An adsorbent was fabricated by immobilizing the above peptide ona porous Sepharose as follows. As the Sepharose, Thiopropyl-Sepharose 6B(Pharmacia LKB) was used. To 50 mg of Thiopropyl-Sepharose 6B was added50 ml of distilled water and the mixture was plated at room temperaturefor 15 minutes to let the resin swell. Then, distilled water was removedand replaced with 0.5 M NaCl-0.1 M Tris-HCl (pH 7.5) coupling buffer.

[0108] On the other hand, 4 mg of the above purified peptide wasdissolved in 400 μL of 0.5 M NaCl-0.1 M Tris-HCl (pH 7.5) couplingbuffer. To this solution was added 150 μL of the above swollenThiopropyl-Sepharose 6B, and the mixture was stirred at 4° C. for 12hours, whereby an adsorbent carrying the purified peptide was obtained.

[0109] This peptide-carrying adsorbent was suction-filtered and thepeptide content in the filtrate was determined by HPLC using theabsolute calibration curve method to find the peptide immobilizationrate. This peptide-carrying adsorbent was washed well with 150 mMNaCl-10 mM phosphate buffer (pH 7.2) and suction-filtered to recoverSepharose 6B-C3Ppt carrying 3.6 mg of the peptide per mL of the carrier.

EXAMPLE 4

[0110] Immobilization of an IgG-Binding Peptide (MK3P47) on a PorousCarrier (Kac)

[0111] Production of MK3P47 Peptide

[0112] A DNA coding for the MK3P47 peptide having the amino acidsequence shown in SEQ ID NO:2 was designed and synthesized so that itcould be ligated to pUCNT Vector (Japanese Kokai PublicationHei-4-212692) by utilizing its Nde I restriction enzyme site for the5′-end and its Hind III restriction enzyme site for the 3′-end. Thenucleotide sequence of the synthesized DNA is shown in SEQ ID NO:3.

[0113] The DNA having the above sequence was ligated to pUCNT Vectorcleaved with the restriction enzymes Nde I and Hind III (Takara Shuzo)in accordance with the manual of Takara Shuzo's DNA Ligation Kit Ver. 2to construct a pUCNTMK3P47 vector (FIG. 3).

[0114] Then, using the known technique, this pUCNTMK3P47 vector DNA wassubcloned in Escherichia coli HB101 (Irivitrogen) and a transformant wasselected with resistance to the antibiotic ampicillin as an indicator.

[0115] From this transformant, the plasmid DNA was extracted andsequenced by the conventional procedure to confirm that it had a DNAsequence conforming to the pUCNTMK3P47 vector design.

[0116] Then, this transformant was shake-cultured in 6L of L-Broth (5g/L NaCl, 10 g/L Bactotrypsin, 5 g/L yeast extract) at 37° C. for 20hours and the cells were recovered by centrifugation (Hitachi RPR9-2rotor, 4° C., 6000 rpm×20 min.).

[0117] The pellet obtained was suspended in 300 mL of TE Buffer (20 mMTris-HC1, 1 mM EDTA; pH 7.5), sonicated (BRANSON 250, in ice, 6 min.×3),and centrifuged (Hitachi RPR16 rotor, 4° C., 15000 rpm×20 min.) and thesupernatant was recovered.

[0118] The above supernatant was heat-treated at 70° C. for 10 minutesand then recentrifuged (Hitachi RPR16 rotor, 4° C., 15000 rpm×20 min.)to provide 300 mL of a supernatant. From this supernatant, the objectiveMK3P47 peptide was isolated as follows. Using a high performance liquidchromatograph (column: Waters'/LBONDASPHERE 5μ C18 300A, 19.0×150 mm),40 ml of acetonitrile was passed at a flow rate of 5 ml/min to activatethe column and, then, 300 mL of the sample was passed at the same flowrate. The column was washed with 200 mL of 0.1% TFA+64% acetonitrile andthe objective MK3P47 peptide was then eluted with 200 mL of 0.1% TFA+40%acetonitrile.

[0119] This fraction was concentrated to 100 mL on an evaporator andlyophilized to provide 1.2 g of a high-purity sample of the peptide.

[0120] Expoxy Activation of Cellulose Gel

[0121] The prototype cellulosic porous rigid gel Kac with a globularprotein cutoff molecular weight of >5×10⁶, prepared by the presentapplicant, 90 mL, was made up with water to 180 mL. Then, 60 mL of 2 Msodium hydroxide was added and the gel temperature was increased to 40°C. To this gel was added 21 mL of epichlorohydrin and the reaction wasconducted at 40° C. with stirring for 1 hour. After completion of thereaction, the gel was thoroughly rinsed with water to provide anepoxy-activated cellulose gel.

[0122] Immobilization of MK3P47 and Determination of the Amount ofImmobilized Protein

[0123] Except that 50 mg of MK3P47 was used in lieu of 4 mg of protein Aand epoxy-activated Kac was used in lieu of epoxy-activated GCL-2000m,the procedure of Example 1 was otherwise repeated to provide Kac-MK3P47(30 mg/mL).

EXAMPLE 5

[0124] Immobilization of an IgG-Binding Peptide (MP47C) on a PorousCarrier (Sephacryl S1000)

[0125] Peptide MP47C having the amino acid sequence shown in SEQ ID NO:4was prepared.

[0126] A DNA (coding for MP47C) of the nucleotide sequence shown in SEQID NO: 5 was designed and synthesized so that it could be ligated topUCNT Vector as in Example 4.

[0127] Thus, a pUCNTMP47C vector was prepared by ligating the above DNAto pUCNT Vector by the same procedure as in Example 4.

[0128] Then, in the same manner as described in Example 4, an E. colitransformant was constructed and from 6L of its culture, 1.3 g of ahigh-purity sample of the objective peptide was obtained and used invarious studies.

[0129] Epoxy Activation of Sephacryl Gel

[0130] The cellulosic porous rigid gel (pore diameter 400 nm) SephacrylS1000 (Pharmacia LKB), 90 mL, was made up with water to 180 mL. Then, 60mL of 2M sodium hydroxide was added and the gel temperature wasincreased to 40° C. To this gel was added 21 mL of epichlorohydrin andthe reaction was conducted at 40° C. with stirring for 1 hour. Aftercompletion of the reaction, the gel was thoroughly rinsed with water toprovide an epoxy-activated Sephacryl gel.

[0131] Immobilization of MP47C and Determination of the Amount ofImmobilized Protein

[0132] Except that 10 mg of MP47C was used in lieu of 4 mg of protein Aand the epoxy-activated Sephacryl gel was used in lieu ofepoxy-activated GCL-2000m, the procedure of Example 1 was otherwiserepeated to provide S1000-MP47C (7 mg/mL).

EXAMPLE 6

[0133] Immobilization of an IgG-Binding Peptide (MP47C) on aSubstantially Nonporous Carrier (Bac)

[0134] Epoxy Activation of Cellulose Gel

[0135] The prototype cellulosic rigid gel nonporous carrier (Bac) with aglobular protein cutoff molecular weight of <3×10⁴, 90 mL, was made upto 180 mL with water. Then, 60 mL of 2 M sodium hydroxide was added andthe gel temperature was increased to 40° C. To this gel was added 21 mLof epichlorohydrin, and the reaction was carried out at 40° C. withstirring for 1 hour. After completion of the reaction, the gel wasthoroughly rinsed with water to provide an epoxy-activated cellulosegel.

[0136] Immobilization of MP47C and Determination of the Amount ofImmobilized Protein

[0137] Except that 30 mg of MP47C was used in lieu of 4 mg of protein Aand epoxy-activated Bac in lieu of epoxy-activated GCL-2000m, theimmobilization procedure of Example 1 was otherwise repeated to provideBac-MP47C (20 mg/mL).

EXAMPLE 7

[0138] Immobilization of a Fragment (Fab) of the Anti-IgG Antibody on aPorous Carrier (CNBr-Activated Sepharose 6B)

[0139] CNBr-activated Sepharose 4B (Pharmacia LKB), 4 g, was swollenwith a small amount of 1 mM HCl/H₂O for 15 minutes and washed with 1 mMHCl/H₂O and coupling buffer (pH 8.3, 0.5 M NaCl, 0.1 M NaHCO₃) in thatorder.

[0140] In 1 mL of coupling buffer was dissolved 1 mg of the Fabavailable on papaine digestion (PIECE, ImmunoPure Fab Preparation Kit)of anti-human IgG (Fab) antibody (Binding Site Co.). To this solutionwas added the above washed gel and the reaction was carried out at 4° C.for 16 hours.

[0141] After the reaction mixture was washed with coupling buffer, blockbuffer (pH 8.3, 0.2 M glycine, 0.5 M NaCl, 0.1 M NaHCO₃) was added andreacted at room temperature for 2 hours.

[0142] The reaction product was washed with two kinds of after-treatmentbuffers (pH 4.0, 0.5 M NaCl, 0.1 M acetic acid-sodium acetate buffer andpH 8.0, 0.5 M NaCl, 0.1 M Tris-HCl buffer) alternately 3 times each toprovide Sepharose 4B-Anti-IgG Fab.

EXAMPLE 8

[0143] Immobilization of the Anti-IgG Antibody on a Porous Carrier(Tresyl Toyopearl)

[0144] The anti-human IgG (Fc) antibody (Binding Site Co.), 1 mg, wasdissolved in 1 mL of coupling buffer (0.5M NaCl, 0.1M carbonate buffer)followed by addition of 200 mg of dry AF-tresyl-Toyopearl 650. Thereaction was carried out at 4° C. overnight.

[0145] After the reaction product was washed with 0.5 M NaCl/water,block buffer (pH 8.0, 0.5 M NaCl, 0.1 M Tris-HCl buffer) was added andreacted at room temperature for 2 hours.

[0146] This reaction mixture was further washed with 0.5 M NaCl/H₂O toprovide Toyopearl-anti-IgG.

EXAMPLE 9

[0147] Evaluation for the Hepatitis C Virus-Adsorbing Performance of theSynthesized Adsorbents

[0148] Adsorption Experiment

[0149] Each adsorbent, 100 μL, was taken in a vial, 100 μl of a patientserum containing hepatitis C virus was added, and the mixture was shakenat 37° C. for 2 hours.

[0150] Determination of Hepatitis C Virus-Adsorbing Capacity

[0151] The above suspension was centrifuged at 5000 rpm for 1 minute andthe amount of hepatitis C virus in the supernatant was determined as HCVRNA (Nippon Roche, Amplicore HCV Monitor).

[0152] As a control experiment, 100 μL of physiological saline in lieuof the adsorbent was taken in a vial and the above procedure wasrepeated to determine the amount of hepatitis C virus in the solution.

[0153] The adsorption rate (%) of hepatitis C virus was calculated bymeans of the following equation.

Adsorption rate (%)=[(Vr−Vt)/Vr]×100

[0154] Vr: concentration of the virus in control solution

[0155] Vt: concentration of the virus in adsorption test supernatant

[0156] The results are shown in Table 1. TABLE 1 HCV RNA adsorption HCVRNA Ad- test control sorption Ex- supernatant solution rate ampleAdsorbent (copy/mL) (copy/mL) (%) 1 GCL2000m-Protein A 2.70E+04 7.90E+0466 2 GCL2000m-Protein G 1.40E+04 7.90E+04 82 3 Sepharose6B-C3Ppt8.10E+04 1.12E+05 28 4 Kac-MK3P47 3.20E+04 1.05E+05 70 5 S1000-MP47C1.00E+04 1.05E+05 91 6 Bac-MP47C 4.20E+04 1.05E+05 60 7 Sepharose4B-4.80E+04 1.05E+05 54 AntiIgGFab 8 Toyopearl-AntiIgG 3.60E+04 1.05E+05 66

EXAMPLE 10

[0157] Evaluation of the Synthesized Adsorbents

[0158] Adsorption Test

[0159] Each adsorbent, 100 μL, was taken in a vial, 100 μL of hepatitisC virus-containing patient serum was added, and the mixture was shakenat 37° C. for 2 hours.

[0160] Determination of High-Density HCV/Low-Density HCV

[0161] The HCV suspension obtained in each adsorption experiment and theHCV suspension obtained using physiological saline in lieu of theadsorbent in otherwise the same manner were respectively admixed withanti-LDL and anti-IgG antibodies and the reaction was carried out of 4°C. for 16 hours. The reaction mixture was centrifuged at 5000 rpm for 15minutes, the pellet was recovered, and the amount of hepatitis C viruswas determined as HCV RNA (Nippon Roche, Amplicore HCV Monitor).Regarding the HCV precipitated by anti-LDL antibody as low-density HCVand the HCV precipitated by anti-IgG antibody as high-density HCV, theHCV RNA ratio (T/B=low-density HCV/high-density HCV) was calculated.

[0162] The results are shown in Table 2. TABLE 2 T/B HCV T/B HCVadsorption control Example Adsorbent experiment experiment 1GCL2000m-Protein A 3/1 1/1  2 GCL2000m-Protein G 2/1 1/1  3Sepharose6B-C3Ppt 3/2 1/1  4 Kac-MK3P47 1/1 1/10 5 S1000-MP47C 1/1 1/106 Bac-MP47C 1/5 1/10 7 Sepharose4B- 1/3 1/10 AntiIgGFab 8Toyopearl-AntiIgG 1/3 1/10

INDUSTRIAL APPLICAPABILITY

[0163] As is clear from the following examples, the present inventionprovides a novel adsorbent having the ability to selectively adsorb andremove the hepatitis C virus present in body fluids and/or the abilityto reduce the ratio of high-density HCV to low-density HCV. Furthermore,by using a body fluid treating apparatus packed with the aboveadsorbent, hepatitis C virus can be selectively removed from body fluidssuch as blood, plasma, and serum.

1 5 1 58 PRT Artificial Sequence lgG - binding domain of protein G 1 CysThr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu 1 5 10 15Thr Thr Thr Lys Ala Val Asp Ala Ala Glu Thr Ala Glu Lys Ala Phe 20 25 30Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp 35 40 45Asp Ala Thr Lys Thr Phe Thr Val Thr Glu 50 55 2 60 PRT ArtificialSequence lgG - binding peptide 2 Met Lys Lys Lys Thr Thr Tyr Lys Leu ValIle Asn Gly Lys Thr Leu 1 5 10 15 Lys Gly Glu Thr Thr Thr Lys Ala ValAsp Ala Glu Thr Ala Glu Lys 20 25 30 Ala Phe Lys Gln Tyr Ala Asn Asp AsnGly Val Asp Gly Val Trp Thr 35 40 45 Tyr Asp Pro Ala Thr Lys Thr Phe ThrVal Thr Glu 50 55 60 3 190 DNA Artificial Sequence DNA coding forSequence ID No 2 3 catatgaaaa agaagaccac ctataaactg gttatcaacggtaaaaccct gaaaggtgaa 60 accaccacca aggctgttga cgctgaaacc gctgaaaaagcatttaaaca gtatgctaac 120 gacaacggtg tcgacggtgt ttggacctat gaccccgctaccaaaacctt taccgttacc 180 gaataagctt 190 4 58 PRT Artificial SequencelgG - binding peptide 4 Met Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys ThrLeu Lys Gly Glu 1 5 10 15 Thr Thr Thr Lys Ala Val Asp Ala Glu Thr AlaGlu Lys Ala Phe Lys 20 25 30 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly ValTrp Thr Tyr Asp Pro 35 40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu Cys 5055 5 184 DNA Artificial Sequence DNA coding for Sequence ID No. 4 5catatgacca cctataaact ggttatcaac ggtaaaaccc tgaaaggtga aaccaccacc 60aaggctgttg acgctgaaac cgctgaaaaa gcatttaaac agtatgctaa cgacaacggt 120gtcgacggtg tttggaccta tgaccccgct accaaaacct ttaccgttac cgaatgctaa 180gctt 184

1. An adsorbent for removing hepatitis C virus which comprises acompound capable of adsorbing hepatitis C virus as immobilized on awater-insoluble carrier.
 2. The adsorbent for removing hepatitis C virusaccording to claim 1 wherein the compound capable of adsorbing hepatitisC virus is a compound having a binding affinity for immunoglobulinand/or immune complex.
 3. An adsorbent for removing hepatitis C viruswhich comprises a compound capable of adsorbing hepatitis C virus boundto immunoglobulin and/or immune complex as immobilized on awater-insoluble carrier.
 4. The adsorbent for removing hepatitis C virusaccording to claim 2 wherein the compound having a binding affinity forimmunoglobulin and/or immune complex is an immunoglobulin-bindingprotein.
 5. The adsorbent for removing hepatitis C virus according toclaim 4 wherein the immunoglobulin-binding protein is at least onemember selected from the group consisting of protein A, protein G,protein H, protein L, protein M, rheumatoid factor, and complement. 6.The adsorbent for removing hepatitis C virus according to claim 2wherein the compound having a binding affinity for immunoglobulin and/orimmune complex is an anti-immunoglobulin antibody.
 7. The adsorbent forremoving hepatitis C virus according to any of claims 1 through 5wherein the compound capable of adsorbing hepatitis C virus is acomponent of an immunoglobulin-binding protein and/oranti-immunoglobulin antibody, which component is a protein fragment or apeptide containing a binding site for immunoglobulin and/or immunecomplex or a derivative of said protein fragment or peptide.
 8. Theadsorbent for removing hepatitis C virus according to any of claims 1through 7 wherein the water-insoluble carrier is a porous carrier. 9.The adsorbent for removing hepatitis C virus according to claim 8wherein the porous carrier has a mean pore diameter ranging 10 from 1500nm.
 10. The adsorbent for removing hepatitis C virus according to any ofclaims 1 through 7 wherein the water-insoluble carrier is asubstantially nonporous carrier.
 11. The adsorbent for removinghepatitis C virus according to any of claims 1 through 10 wherein thewater-insoluble carrier is a hydrophilic carrier.
 12. The adsorbent forremoving hepatitis C virus according to any of claims 1 through 11 whichis used for removing hepatitis C virus from a body fluid such as blood,plasma, or the like.
 13. The adsorbent for removing hepatitis C virusaccording to any of claims 1 through 12 wherein the hepatitis C virus isan immune complex virus.
 14. An apparatus for adsorbing hepatitis Cvirus which comprises a casing having an inlet and an outlet foradmission and discharge of a fluid and housing the adsorbent forremoving hepatitis C virus according to any of claims 1 through 13, anda means for preventing leakage of said adsorbent for removing hepatitisC virus from the casing.
 15. A method for adsorbing hepatitis C viruswhich comprises a step of contacting the adsorbent for removinghepatitis C virus according to any of claims 1 through 13 with a fluidcontaining hepatitis C virus.
 16. The method for adsorbing hepatitis Cvirus according to claim 15 wherein the fluid containing hepatitis Cvirus is blood, plasma or like body fluids.